Novel hydrogen sulfate salt

ABSTRACT

The present invention relates to Compound 1 hydrogen sulfate salt and solvates, crystalline forms and amorphous forms thereof, and to processes for their preparation.

RELATED APPLICATION

The present application is a divisional application of U.S. applicationSer. No. 12/097,942, filed Oct. 14, 2008, which claims priority of U.S.Provisional Application Ser. No. 60/752,781 filed Dec. 21, 2005, each ofwhich is incorporated herein in its entirety by this reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel salt and, more particularly, toa novel salt of6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide (hereinafter referred to as “Compound 1”),which is a MEK inhibitor that is useful in the treatment and/orprophylaxis of proliferative disease states, such as cancer, in amammal. More specifically, the present invention relates to a hydrogensulfate salt of Compound 1 and to processes for the preparation of saidsalt. Also provided are pharmaceutical compositions containing ahydrogen sulfate salt of Compound 1, and the use of the salt in themanufacture of medicaments for treatment and/or prophylaxis ofproliferative disease states, such as cancer, in the human or animalbody, and methods of treating proliferative disease states, such ascancer, in a mammal by administering a therapeutically effective amountof a hydrogen sulfate salt of Compound 1.

2. Description of the State of the Art

Cell signaling through growth factor receptors and protein kinases is animportant regulator of cell growth, proliferation and differentiation.In normal cell growth, growth factors, through receptor activation(i.e., PDGF or EGF and others), activate MAP kinase pathways. One of themost important and most well understood MAP kinase pathways involved innormal and uncontrolled cell growth is the Ras/Raf kinase pathway.Active GTP-bound Ras results in the activation and indirectphosphorylation of Raf kinase. Raf then phosphorylates MEK1 and 2 on twoserine residues (S218 and S222 for MEK1 and S222 and S226 for MEK2) (Ahnet al., Methods in Enzymology, 2001, 332:417-431). Activated MEK thenphosphorylates its only known substrates, the MAP kinases ERK1 and 2.ERK phosphorylation by MEK occurs on Y204 and T202 for ERK1 and Y185 andT183 for ERK2 (Ahn et al., Methods in Enzymology 2001, 332:417-431).Phosphorylated ERK dimerizes and then translocates to the nucleus whereit accumulates (Khokhlatchev et al., Cell 1998, 93:605-615). In thenucleus, ERK is involved in several important cellular functions,including but not limited to nuclear transport, signal transduction, DNArepair, nucleosome assembly and translocation, and mRNA processing andtranslation (Ahn et al., Molecular Cell, 2000, 6:1343-1354). Overall,treatment of cells with growth factors leads to the activation of ERK1and 2 which results in proliferation and, in some cases, differentiation(Lewis et al., Adv. Cancer Res. 1998, 74: 49-139).

In proliferative diseases, genetic mutations and/or overexpression ofthe growth factor receptors, downstream signaling proteins, or proteinkinases involved in the ERK kinase pathway lead to uncontrolled cellproliferation and, eventually, tumor formation. For example, somecancers contain mutations which result in the continuous activation ofthis pathway due to continuous production of growth factors. Othermutations can lead to defects in the deactivation of the activatedGTP-bound Ras complex, again resulting in activation of the MAP kinasepathway. Mutated, oncogenic forms of Ras are found in 50% of colonand >90% pancreatic cancers as well as many others types of cancers(Kohl et al., Science, 1993, 260:1834-1837). Recently, bRaf mutationshave been identified in more than 60% of malignant melanoma (Davies, H.,et al., Nature 2002, 417:949-954). These mutations in bRaf result in aconstitutively active MAP kinase cascade. Studies of primary tumorsamples and cell lines have also shown constitutive or overactivation ofthe MAP kinase pathway in cancers of pancreas, colon, lung, ovary andkidney (Hoshino, R., et al., Oncogene 1999, 18:813-822). Hence, there isa strong correlation between cancers and an overactive MAP kinasepathway resulting from genetic mutations.

As constitutive or overactivation of MAP kinase cascade plays a pivotalrole in cell proliferation and differentiation, inhibition of thispathway is believed to be beneficial in hyperproliferative diseases. MEKis a key player in this pathway as it is downstream of Ras and Raf.Additionally, it is an attractive therapeutic target because the onlyknown substrates for MEK phosphorylation are the MAP kinases, ERK1 and2. Inhibition of MEK has been shown to have potential therapeuticbenefit in several studies. For example, small molecule MEK inhibitorshave been shown to inhibit human tumor growth in nude mouse xenografts,(Sebolt-Leopold et al., Nature-Medicine 1999, 5(7):810-816; Trachet etal., AACR Apr. 6-10, 2002, Poster #5426; Tecle, H., IBC 2^(nd)International Conference of Protein Kinases, Sep. 9-10, 2002), blockstatic allodynia in animals (WO 01/05390) and inhibit growth of acutemyeloid leukemia cells (Milella et al., J. Clin. Invest. 2001, 108(6):851-859).

Small molecule inhibitors of MEK have been disclosed. At least thirteenpatent applications have appeared in the last several years: U.S. Pat.No. 5,525,625; WO 98/43960; WO 99/01421; WO 99/01426; WO 00/41505; WO00/42002; WO 00/42003; WO 00/41994; WO 00/42022; WO 00/42029; WO00/68201; WO 01/68619; and WO 02/06213.

Inhibitors of the MEK are also described in WO 03/077914.6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide, or “Compound 1”, is exemplified in WO03/077914 and possesses the following structural formula:

Compound 1 has been shown to possess inhibitory activity against MEK andtherefore to be useful in the treatment of a hyperproliferative diseasesuch as cancer.

WO 03/077914 discloses, in general terms, certain pharmaceuticallyacceptable salts of the compounds disclosed therein. Specifically, it isstated in WO 03/077914 that pharmaceutically acceptable salts of thecompounds disclosed therein that possess a sufficiently basic moiety mayform acid addition salts containing pharmaceutically acceptable anions,and a range of such anions are listed. Similarly, suitable salts of thecompounds possessing an acidic moiety are to be formed by treatment of acompound with a basic compound and particularly an inorganic base.

The form of a pharmaceutically active compound which is used inmedicaments is suitably one that provides for reasonable handlingproperties, which allow it to be processed and formulated. However, itis also necessary to ensure that the biological properties of the finalformulation, such as dissolution rate of tablets and bioavailability ofactive ingredient are optimized, and there is frequently compromises tobe made in selecting a particular form which best fulfils all thesevarious requirements. However, in some cases, salts do not form easilyand/or are not stable, which is probably due to low pKa values. The pKavalue expresses the strength of acids and base, i.e., the tendency foran acid to lose a proton or a base to add a proton (Bronsted J. N., Rec.Tray. Chim. (1923) 47:718). This is particularly true for Compound 1.

SUMMARY OF THE INVENTION

The present invention provides a hydrogen sulfate salt (1:1 drug:H₂SO₄)of Compound 1 and various forms thereof, all of which are includedwithin the scope of the invention. The salt may be in various forms, allof which are included within the scope of the invention. These formsinclude anhydrous forms as well as solvates. A further form may beproduced by desolvating solvates. In a particular embodiment the salt isin the anhydrous form.

In a further aspect the present invention provides a method of using ahydrogen sulfate salt of Compound 1 as a medicament to treat ahyperproliferative disease or condition.

An additional aspect of the invention is the use of a hydrogen sulfatesalt of Compound 1 in the preparation of a medicament for the treatmentor prevention of a hyperproliferative disease or condition.

Additional advantages and novel features of this invention shall be setforth in part in the description that follows, and in part will becomeapparent to those skilled in the art upon examination of the followingspecification or may be learned by the practice of the invention. Theadvantages of the invention may be realized and attained by means of theinstrumentalities, combinations, compositions, and methods particularlypointed out in the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated herein and form a partof the specification, illustrate non-limiting embodiments of thisinvention, and together with the description, serve to explain theprinciples of the invention.

In the Figures:

FIG. 1 shows the XRPD of the hydrogen sulfate salt of Compound 1;

FIG. 2 shows the infrared spectrum of the hydrogen sulfate salt ofCompound 1 obtained using the DRIFTS sampling technique; and

FIG. 3 shows the results of plasma concentration levels of Compound 1following administration of 150 mg free base equivalent oral dispersiondoses of Compound 1 (x) and the hydrogen sulfate salt to fasted dogs(▴).

DETAILED DESCRIPTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents, which may be included within the scopeof the present invention as defined by the claims. One skilled in theart will recognize many methods and materials similar or equivalent tothose described herein, which could be used in the practice of thepresent invention. The present invention is in no way limited to themethods and materials described. In the event that one or more of theincorporated literature, patents, and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The present invention provides a hydrogen sulfate salt (1:1 drug toH₂SO₄) of Compound 1 and various forms thereof, all of which areincluded within the scope of the invention. The salt may be in variousforms, all of which are included within the scope of the invention.These forms include anhydrous forms as well as solvates. A further formmay be produced by desolvating solvates. In a particular embodiment, thesalt is anhydrous hydrogen sulfate salt of Compound 1. Further, thepresent invention provides a hydrogen sulfate salt form of Compound 1which shows unique physical and pharmaceutical properties that make itparticularly suitable for use in medicaments.

In certain embodiments, salts of Compound 1 are crystalline. Thecrystalline salts have been found to be better than the free base interms in their handling properties from a manufacturing point of view,in particular their static and flow properties. The formation of saltsmay provide a means of purification, as process impurities can beseparated and salts are generally easier to isolate than the free base.

In one embodiment of the invention the hydrogen sulfate salt of Compound1 is a crystalline salt, which has surprisingly been found to possessimproved pharmaceutical properties when compared to the free base ofCompound 1 and certain other salt forms of Compound 1. In particular,the dissolution rate of this crystalline salt, as well as itsbioavailability has been found to be particularly high as compared tothe free base and other salts, as illustrated in the exampleshereinafter. The enhanced bioavailability of the hydrogen sulfate saltof Compound 1 over the free base has been shown to be independent of theformulation used for administration. The bioavailability of the freebase and hydrogen sulfate forms have been compared herein when dosed inthe same dispersion formulations, but similar differences inbioavailability were also observed for simple tablet formulations.

When it is stated that the present invention relates to a salt ofCompound 1 which is a crystalline salt the degree of crystallinity isconveniently greater than about 60%, more conveniently greater thanabout 80%, preferably greater than about 90% and more preferably greaterthan about 95%. Most preferably the degree of crystallinity is greaterthan about 98%.

The extent of enhanced bioavailability offered by the hydrogen sulfatesalt is surprising and is particularly useful, since the free base ofCompound 1 has been classified as a BCS Class 4 compound. BCS Class 4compounds normally have low bioavailability due to both low dissolutionrate and permeability, and the limitation of permeability on absorptionmeans that such salts would not usually be expected exert a substantialimpact on absorption (See for example: Dressman et al. (2001) PharmTech. July: 68).

Suitable solvates of the hydrogen sulfate salt of Compound 1 are formedfrom a wide range of solvents, in particular organic solvents such astetrahydrofuran (THF), acetonitrile (ACN), ethanol (EtOH) and methanol(MeOH). Suitable organic solvents include esters such as C₁₋₆ alkylesters, for example ethyl acetate, and ketones such as C₁₋₆ alkylketones, for example methyl ethyl ketone (2-butanone).

Preparation of the salt can be effected by reacting a slurry of Compound1 in an organic solvent and water with sulfuric acid. For preparation ofa 1:1 salt approximately 1 equivalent of sulfuric acid is used. Thus ina further aspect, the invention provides a method for preparing ahydrogen sulfate salt of Compound 1, said method comprising:

(i) reacting a slurry of Compound 1 in an organic liquid and water withapproximately 1 equivalent of sulfuric acid;

(ii) recovering the salt from the resultant solution; and

(iii) thereafter, if desired or necessary, forming a solvate thereof.

The mole ratio of the amount of sulfuric acid to Compound 1 is suitablyin the range of from 1.00:1 to 2:1, for example in a range from 1.05:1to 1.15:1. The sulfuric acid used is suitably in the form ofconcentrated sulfuric acid. In a particularly embodiment, the mole ratioof sulfuric acid to Compound 1 is 1.10:1.0.

Suitably the amount of water added in step (i) is restricted to thatnecessary to ensure that the salt is formed. The precise amounts usedwill depend upon the particular nature of the solvent, the concentrationof the sulfuric acid etc., but typically, the water will be present inan amount of less than 20% v/v of the total liquid present, for examplefrom 13-17% v/v.

In a particular embodiment, the organic solvent used in step (i) is2-butanone (methyl ethyl ketone), water is approximately 15% of theliquid volume, and the total amount of liquid used relative to Compound1 is approximately 8 mL per gram of Compound 1.

Suitably, addition of sulfuric acid in step (i) is carried out in acontrolled manner, for example at a temperature below 10° C., and theremainder of step (i) is then carried out at elevated temperature, forexample from 30-90° C., as a further example in a range between 55-75°C., and as a further example at about 65° C.

Suitable organic liquids include organic solvents in which Compound 1and its salts are sparingly soluble. As used herein, the expression“sparingly soluble” means having a solubility less than 100 mL ofsolvent per gram of solute, for example between 30 and 100 mL of solventper gram of solute. These solvents include alkyl ketones, for exampleC₁₋₆ alkyl ketones such as 2-butanone, alcohols such as C₁₋₆ alcohols,for example methanol or ethanol, and esters such as C₁₋₆ alkyl esters,for example as ethyl acetate. In one embodiment, the organic solvent ismethyl ethyl ketone (2-butanone).

Suitably, the reaction mixture is filtered between steps (i) and (ii) toremove any extraneous material. The residue is optionally washed, forexample with a mixture of the organic liquid and water, and the desiredsalt crystallized from the filtrate, which may optionally be combinedwith the wash solution.

In certain embodiments, the hydrogen sulfate salt is recovered in step(ii) by cooling the reaction mixture, optionally with the addition offurther organic liquid, so that the hydrogen sulfate salt isprecipitated. The further organic liquid may be the same organic liquidas used in step (i), or it may be a different organic liquid, providedthis acts as an anti-solvent for the hydrogen sulfate salt ofCompound 1. Seeding of the solution with crystals of the hydrogensulfate salt of Compound 1 may assist in the precipitation process.

In one embodiment, prior to cooling, the filtrate is first subjected toa distillation step to remove water and to ensure that the salt isrecovered in an acceptable yield. In a particular embodiment the solventis 2-butanone and the filtrate is distilled at atmospheric pressure.

On cooling, the salt can be recovered from the resultant slurry forexample by filtration. The recovered material may then be dried forexample at elevated temperature, for example of from 40-60° C., and asanother example at about 50° C., until constant weight is achieved. Ifthe product is a solvate with the organic liquid such as methanol, itmay be de-solvated if desired at this time by heating.

The physical properties of the hydrogen sulfate salt were investigatedand are described further in the Examples.

The invention also includes isotopically-labeled compounds, which areidentical to those recited in the present invention, but for the factthat one or more atoms are replaced by an atom having an atomic mass ormass number different from the atomic mass or mass number usually foundin nature. Examples of isotopes that can be incorporated into compoundsof the invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, sulfur, fluorine and chloride, such as ²H, ³H, ¹³C, ¹⁴C,¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶Cl, respectively. The hydrogensulfate salt of Compound 1 and polymorphs thereof which contain theaforementioned isotopes and/or other isotopes of other atoms are withinthe scope of this invention. Certain isotopically-labeled compounds ofthe present invention, for example those into which radioactive isotopessuch as ³H and ¹⁴C are incorporated, are useful in drug and/or substratetissue distribution assays. Tritiated, i.e., ³H and carbon-14, i.e.,¹⁴C, isotopes are particularly widely used as a result of their ease ofpreparation and detectability. Further, substitution with heavierisotopes such as deuterium, i.e., ²H, can afford certain therapeuticadvantages resulting from greater metabolic stability, for exampleincreased in vivo half-life or reduced dosage requirements and, hence,may be utilized in some particular circumstances. Isotopically labeledsalts of the present invention can generally be prepared by carrying outprocedures disclosed in WO 03/077914 by substituting a readily availableisotopically labeled reagent for a non-isotopically labeled reagentduring the preparation, or if desired, using an isotopically labeledsulfuric acid in the preparation of the salt.

The composition may be in a form suitable for oral administration (forexample as tablets, lozenges, hard or soft capsules, emulsions,dispersible powders or granules, syrups, elixirs or oily orextemporaneously prepared aqueous suspensions), for administration byinhalation (for example as a finely divided powder or a liquid aerosol),for administration by insufflation (for example as a finely dividedpowder), for parenteral injection (for example as a sterile solution,suspension or emulsion for intravenous, subcutaneous, intramuscular,intravascular or infusion dosing), for topical administration (forexample as creams, ointments, gels, oily solutions or suspensions orextemporaneously prepared aqueous suspensions), or for rectaladministration (for example as a suppository). In one embodiment, thehydrogen sulfate salt of Compound 1 is administered orally. In generalthe above compositions may be prepared in a conventional manner usingconventional excipients.

The amount of the active compound administered will be dependent on thesubject being treated, the severity of the disorder or condition, therate of administration, the disposition of the compound and thediscretion of the prescribing physician. However, an effective dosage isin the range of about 0.01 to about 100 mg per kg body weight per day,preferably about 1 to about 35 mg/kg/day, in single or divided doses.For a 70 kg human, this would amount to about 0.7 to 7000 mg/day,preferably about 70 to about 2500 mg/day. In some instances, dosagelevels below the lower limit of the aforesaid range may be more thanadequate, while in other cases still larger doses may be employedwithout causing any harmful side effect, provided that such larger dosesare first divided into several small doses for administration throughoutthe day. A unit dosage form such as a tablet or capsule will usuallycontain, for example 1-1000 mg of active ingredient, and preferably5-420 mg of active ingredient. Preferably a daily dose in the range of0.03-6 mg/kg is employed.

According to a further aspect of the present invention there is provideda hydrogen sulfate salt of Compound 1 as defined herein for use in amethod of treatment or prophylaxis of the human or animal body bytherapy. A further feature of the present invention is hydrogen sulfatesalt of Compound 1 as defined herein for use as a medicament. In afurther aspect, the present invention provides hydrogen sulfate salt ofCompound as defined herein for use as a medicament for the treatment ofdisease states mediated through MEK, in particular proliferativedisorders, or abnormal cell growth, such as cancer, in a warm-bloodedmammal such as a human. Accordingly, a further aspect of the inventionprovides the use of hydrogen sulfate salt of Compound 1 as definedherein in the manufacture of a medicament for use in the treatment ofdisease states mediated through the MEK, in particular proliferativedisorders, or abnormal cell growth, such as cancer, in a warm-bloodedmammal such as a human.

According to a further feature of the invention there is provided amethod for treating disease states mediated through the MEK, inparticular proliferative disorders, or abnormal cell growth, such ascancer, in a warm-blooded mammal, such as a human, in need of suchtreatment which comprises administering to said mammal an effectiveamount of an hydrogen sulfate salt of Compound 1 hydrogen sulfate saltas herein, or a pharmaceutical composition as defined herein.

Particular examples of proliferative disorders, which may be treatedusing the salts or compositions of the invention, includehyperproliferative disorders in a mammal Particular cancers are brain,lung, squamous cell, bladder, gastric, pancreatic, breast, head, neck,renal, kidney, ovarian, prostate, colorectal, esophageal, testicular,gynecological or thyroid cancer.

However, the compounds and compositions of the invention may also beused in the treatment of a non-cancerous hyperproliferative disordersuch as benign hyperplasia of the skin (e.g., psoriasis), restenosis, orprostate (e.g., benign prostatic hypertrophy (BPH)).

Other examples of MEK mediated diseases, which may be treated using thecompounds, or compositions of the invention include pancreatitis orkidney disease (including proliferative glomerulonephritis anddiabetes-induced renal disease) or the treatment of pain in a mammal.

The compounds and compositions may also be used for the prevention ofblastocyte implantation in a mammal, or for treating a disease relatedto vasculogenesis or angiogenesis in a mammal. Such diseases may includetumor angiogenesis, chronic inflammatory disease such as rheumatoidarthritis, atherosclerosis, inflammatory bowel disease, skin diseasessuch as psoriasis, eczema, and scleroderma, diabetes, diabeticretinopathy, retinopathy of prematurity, age-related maculardegeneration, hemangioma, glioma, melanoma, Kaposi's sarcoma andovarian, breast, lung, pancreatic, prostate, colon and epidermoidcancer.

The terms “abnormal cell growth” and “hyperproliferative disorder” areused interchangeably in this application and refer to cell growth thatis independent of normal regulatory mechanisms (e.g., loss of contactinhibition). This includes, for example, the abnormal growth of: (1)tumor cells (tumors) that proliferate by expressing a mutated tyrosinekinase or over expression of a receptor tyrosine kinase; (2) benign andmalignant cells of other proliferative diseases in which aberranttyrosine kinase activation occurs; (3) any tumors that proliferate byreceptor tyrosine kinases; (4) any tumors that proliferate by aberrantserine/threonine kinase activation; and (5) benign and malignant cellsof other proliferative diseases in which aberrant serine/threoninekinase activation occurs.

The term “treating”, as used herein, unless otherwise indicated, meansreversing, alleviating, inhibiting the progress of, or preventing thedisorder or condition to which such term applies, or one or moresymptoms of such disorder or condition. The term “treatment” as usedherein, unless otherwise indicated, refers to the act of treating as“treating” is defined immediately above.

Thus patients that can be treated with compounds or compositions of thepresent invention include, for example, patients that have beendiagnosed as having psoriasis, restenosis, atherosclerosis, BPH, lungcancer, non small cell lung cancer, bone cancer, CMML, pancreaticcancer, colorectal, skin cancer, cancer of the head and neck, melanoma(in particular cutaneous or intraocular melanoma), uterine cancer,ovarian cancer, rectal cancer, cancer of the anal region, stomachcancer, colon cancer, breast cancer, testicular, gynecologic tumors(e.g., uterine sarcomas, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina orcarcinoma of the vulva), ovarian cancer, multiple myeloma,hepatocellular carcinoma, Hodgkin's disease, cancer of the esophagus,cancer of the small intestine, cancer of the endocrine system (e.g.,cancer of the thyroid, parathyroid or adrenal glands), sarcomas of softtissues, cancer of the urethra, cancer of the penis, prostate cancer,chronic or acute leukemia, in particular acute myeloid leukaemia solidtumors of childhood, lymphocytic lymphomas, cancer of the bladder,cancer of the kidney or ureter (e.g., renal cell carcinoma, carcinoma ofthe renal pelvis), or neoplasms of the central nervous system (e.g.,primary CNS lymphoma, spinal axis tumors, brain stem gliomas orpituitary adenomas).

The hydrogen sulfate salt of Compound 1 may be applied as a sole therapyor may involve, in addition to the hydrogen sulfate salt of Compound 1,one or more other substances and/or treatments. Such conjoint treatmentmay be achieved by way of the simultaneous, sequential or separateadministration of the individual components of the treatment. In thefield of medical oncology it is normal practice to use a combination ofdifferent forms of treatment to treat each patient with cancer. Inmedical oncology the other component(s) of such conjoint treatment inaddition to Compound 1 hydrogen sulfate salt may be surgery,radiotherapy or chemotherapy. Such chemotherapy may cover categories oftherapeutic agent such as:

(i) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, (for example the anti-vascularendothelial cell growth factor antibody bevacizumab [Avastin™], and VEGFreceptor tyrosine kinase inhibitors such as4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO98/35985) and SU11248 (sunitinib; WO 01/60814), compounds such as thosedisclosed in International Patent Applications WO97/22596, WO 97/30035,WO 97/32856 and WO 98/13354 and those that work by different mechanismsfrom those defined herein (for example linomide, inhibitors of integrinαvβ3 function, angiostatin, razoxin, thalidomide, MMP-2(matrix-metalloprotienase 2) inhibitors, MMP-9 (matrix-metalloprotienase9) inhibitors, and COX-II (cyclooxygenase II) inhibitors), and

(ii) vascular targeting agents (for example combretastatin phosphate andcompounds disclosed in WO 00/40529, WO 00/41669, WO 01/92224, WO02/04434 and WO 02/08213, and the vascular damaging agents described inInternational Patent Application Publication No. WO 99/02166, (forexample N-acetylcolchinol-O-phosphate));

(iii) cytostatic agents such as antioestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene, and iodoxyfene), oestrogen receptordown regulators (for example fulvestrant), progestogens (for examplemegestrol acetate), aromatase inhibitors (for example anastrozole,letrazole, vorazole, and exemestane), antiprogestogens, antiandrogens(for example flutamide, nilutamide, bicalutamide, and cyproteroneacetate), LHRH agonists and antagonists (for example goserelin acetate,leuprorelin, and buserelin), inhibitors of 5α-reductase (for examplefinasteride);

(iv) anti-invasion agents (for example metalloproteinase inhibitors likemarimastat and inhibitors of urokinase plasminogen activator receptorfunction or antibodies to Heparanese;

(v) inhibitors of growth factor function (such growth factors includefor example platelet derived growth factor and hepatocyte growthfactor), such inhibitors include growth factor antibodies, growth factorreceptor antibodies, (for example the anti-erbb2 antibody trastuzumab[Herceptin™], the anti-EGFR antibody panitumumab, the anti-erbB1antibody cetuximab [C225]), and any growth factor or growth factorreceptor antibodies disclosed by Stern et al. Critical reviews inoncology/haematology, 2005, Vol. 54, pp 11-29); such inhibitors alsoinclude tyrosine kinase inhibitors such as inhibitors of the epidermalgrowth factor family (for example EGFR family tyrosine kinase inhibitorssuch asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)-quinazolin-4-amine(gefitinib, AZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine (CI 1033)) and erbB2 tyrosine kinase inhibitors suchas lapatinib, inhibitors of the hepatocyte growth factor family,inhibitors of the platelet-derived growth factor family such asimatinib, inhibitors of serine/threonine kinases (for example Ras/Rafsignalling inhibitors such as farnesyl transferase inhibitors, forexample sorafenib (BAY 43-9006)), inhibitors of cell signalling throughMEK and/or AKT kinases, c-kit inhibitors, abl kinase inhibitors, IGFreceptor (insulin-like growth factor) kinase inhibitors; aurora kinaseinhibitors (for example AZD1152, PH739358, VX-680, MLN8054, R763, MP235,MP529, VX-528 AND AX39459) and cyclin dependent kinase inhibitors suchas CDK2 and/or CDK4 inhibitors;

(vi) antiproliferative/antineoplastic drugs and combinations thereof, asused in medical oncology, such as antimetabolites (for exampleantifolates such as methotrexate, fluoropyrimidines such as5-fluorouracil, tegafur, purine and adenosine analogues, and cytosinearabinoside, hydroxyurea or, for example, one of the anti-metabolitesspecifically disclosed in European Patent Application No. 239362 such asN-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino-2-thenoyl)-L-glutamicacid; antitumour antibiotics (for example anthracyclines such asadriamycin, bleomycin, doxorubicin, daunomycin, epirubicin andidarubicin, mitomycin-C, dactinomycin, and mithramycin); platinumderivatives (for example cisplatin, and carboplatin); alkylating agents(for example nitrogen mustard, melphalan, chlorambucil, busulphan,cyclophosphamide, ifosfamide, nitrosoureas, and thiotepa); antimitoticagents (for example vinca alkaloids such as vincristine, vinblastine,vindesine, and vinorelbine, and taxoids such as taxol and taxotere);topoisomerase inhibitors (for example epipodophyllotoxins such asetoposide and teniposide, amsacrine, topotecan, camptothecin andirinotecan); enzymes (for example asparaginase); and thymidylatesynthase inhibitors (for example raltitrexed);

and additional types of chemotherapeutic agent including:

(vii) biological response modifiers (for example interferon);

(viii) antibodies (for example edrecolomab);

(ix) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense;

(x) gene therapy approaches, including for example approaches to replaceaberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT(gene-directed enzyme pro-drug therapy) approaches such as those usingcytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi-drug resistance gene therapy; and

(xi) immunotherapy approaches, including for example ex-vivo and in vivoapproaches to increase the immunogenicity of patient tumour cells, suchas transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte-macrophage colony stimulating factor, approaches to decreaseT-cell anergy, approaches using transfected immune cells such ascytokine-transfected dendritic cells, approaches usingcytokine-transfected tumour cell lines and approaches usinganti-idiotypic antibodies.

For example, a hydrogen sulfate salt of Compound 1 may be used inconjunction with an effective amount of one or more substances selectedfrom anti-angiogenesis agents, signal transduction inhibitors, andantiproliferative agents.

In a particular embodiment, anti-angiogenesis agents, such as MMP-2(matrix-metalloprotienase 2) inhibitors, MMP-9 (matrix-metalloprotienase9) inhibitors, and COX-II (cyclooxygenase II) inhibitors, can be used inconjunction with a Compound 1 hydrogen sulfate salt of the presentinvention and pharmaceutical compositions described herein. Examples ofuseful COX-II inhibitors include CELEBREX™ (alecoxib), valdecoxib, androfecoxib. Examples of useful matrix metalloprotienase inhibitors aredescribed in WO 96/33172, WO 96/27583, WO 98/07697, WO 98/03516, WO98/34918, WO 98/34915, WO 98/33768, WO 98/30566, WO 90/05719, WO99/52910, WO 99/52889, WO 99/29667, U.S. Pat. No. 5,863,949, and U.S.Pat. No. 5,861,510, all of which are incorporated herein in theirentireties by reference. Suitable MMP-2 and MMP-9 inhibitors are thosethat have little or no activity inhibiting MMP-1. In particular, thosethat selectively inhibit MMP-2 and/or MMP-9 relative to the othermatrix-metalloproteinases (i.e., MMP-1, MMP-3, MMP-4, MMP-5, MMP-6,MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13) are used. Particularexamples of MMP inhibitors useful in the present invention are AG-3340,RO 32-3555, and RS 13-0830.

Therefore, a further aspect of the present invention the hydrogensulphate salt of Compound 1 in combination with any one of the antitumour agents listed under (i)-(xi) herein above. A further aspect ofthe present invention provides the hydrogen sulphate salt of Compound 1in combination with one or more of the anti tumour agents listed under(i)-(xi) herein above. A further aspect of the present inventionprovides the hydrogen sulphate salt of Compound 1 in combination withany one of the classes of anti-tumour agents listed under (i)-(xi)herein above.

Herein, where the term “combination” is used it is to be understood thatthis refers to simultaneous, separate or sequential administration. Inone aspect of the invention “combination” refers to simultaneousadministration. In another aspect of the invention “combination” refersto separate administration. In a further aspect of the invention“combination” refers to sequential administration. Where theadministration is sequential or separate, the delay in administering thesecond component should not be such as to lose the beneficial effect ofthe combination.

According to a further aspect of the present invention there is provideda kit comprising the hydrogen sulphate salt of Compound 1 in combinationwith an anti-tumour agent selected from one listed under (i)-(xi) hereinabove.

According to a further aspect of the present invention there is provideda kit comprising:

a) the hydrogen sulphate salt of Compound 1 in a first unit dosage form;

b) an anti-tumour agent selected from one listed under (i)-(xi) hereinabove; in a second unit dosage form; and

c) container means for containing said first and second dosage forms.

Compound 1 has been found to have activity in the following assay.N-terminal 6 His-tagged, constitutively active MEK1 (2-393) is expressedin E. coli and protein is purified by conventional methods (Ahn et al.,Science 1994, 265:966-970). The activity of MEK1 is assessed bymeasuring the incorporation of γ-³³P-phosphate from γ-³³P-ATP ontoN-terminal His tagged ERK2, which is expressed in E. coli and ispurified by conventional methods, in the presence of MEK1. The assay iscarried out in 96-well polypropylene plate. The incubation mixture (100μL) comprises of 25 mM Hepes, pH 7.4, 10 mM MgCl₂, 5 mMβ-glycerolphosphate, 100 μM sodium orthovanadate, 5 mM DTT, 5 nM MEK1,and 1 μM ERK2. Inhibitors are suspended in DMSO, and all reactions,including controls are performed at a final concentration of 1% DMSO.Reactions are initiated by the addition of 10 μM ATP (with 0.5 μCiγ-³³P-ATP/well) and incubated at ambient temperature for 45 minutes.Equal volume of 25% TCA is added to stop the reaction and precipitatethe proteins. Precipitated proteins are trapped onto glass fiber Bfilterplates, and excess labeled ATP washed off using a Tomtec MACH IIIharvestor. Plates are allowed to air-dry prior to adding 30 μL/well ofPackard Microscint 20, and plates are counted using a Packard TopCount.In this assay, Compound 1 exhibited an IC₅₀ of less than 50 micromolar.

EXAMPLES

In order to illustrate the invention, the following examples areincluded. However, it is to be understood that these examples do notlimit the invention and are only meant to suggest a method of practicingthe invention. Yields are given for the Examples as performed and couldlikely be enhanced through further development. The ¹H NMR spectrum (400MHz) was referenced to TMS (0.00 ppm), ¹³C NMR spectrum (100 MHz) wasreferenced to the NMR solvent (39.5 ppm) and the ¹⁹F NMR spectrum wasreferenced to trichlorofluoromethane (0.00 ppm). FTIR spectra wereobtained on a Nicolet Magna 860 ESP FTIR Spectrometer in various waysincluding from a 2% w/w dispersion of this material in powdered KBr,using the DRIFTS sampling technique, over the 4,000-400 cm⁻¹mid-infrared spectral region.

Example 1 Preparation of the Hydrogen Sulfate Salt of Compound 1

To a stirred suspension of6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide (100 g, 0.206 mol) (obtainable asdescribed in Example 10 of WO 03/077914, which is incorporated herein byreference and as described below) in 2-butanone (680 mL) and water (115mL) at 0-5° C. was added sulfuric acid (12.3 mL, 0.226 mol) followed bywater (5 mL) maintaining a temperature of 10° C. or lower. The stirredmixture was heated to 65° C. and held for 30 minutes before filtering toremove any extraneous matter. The filter was washed with a mixture of2-butanone (85 mL) and water (15 mL). The combined filtrates were heatedto 72° C. before adding 2-butanone (500 mL) maintaining a temperature ofbetween 60-72° C. The resulting mixture was distilled at atmosphericpressure (approximate distillation temperature 73-74° C.) until 500 mLof distillate had been collected.

A second aliquot of 2-butanone (500 mL) was added, maintaining thetemperature of the mixture above 70° C. The resulting mixture wasdistilled again until 250 mL of distillate had collected. The mixturewas cooled to 0-5° C. over approximately 1 hour. The resulting slurrywas filtered, washed with 2-butanone (240 mL) and dried under reducedpressure at 50° C., until a constant weight was achieved, to give6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide hydrogen sulfate (103.5 g, 0.186 mol, 90%yield) as an off white crystalline solid. ¹H NMR (400 MHz, D₆ DMSO) δ3.58 (2H, t, CH₂OH), 3.89 (2H, t, CH₂ON), 3.99 (3H, s, CH₃), 6.47 (1H,dd, ArH), 7.29 (1H, dd, ArH), 7.63 (1H, d, ArH), 7.91 (1H, s, ArH), 7.96(3H, br, ROH, NH, SOH), 8.10 (1H, br, ArNH), 8.94 (1H, s, NCHN), 11.79(1H, s, ONH). ¹³C NMR (100 MHz, D₆ DMSO) δ 32.1 (CH₃), 58.5 (CH₂OH),77.3 (CH₂ON), 108.2 (CH), 109.6 (CBr), 115.8 (CH), 120.6 (CCl), 122.0(C), 125.0 (CC═O), 129.4 (C), 130.5 (CH), 131.1 (CH), 132.3 (C), 140.6(C), 145.8 (CF), 146.5 (CH), 164.2 (C═O).

The results of the infrared analysis are shown in FIG. 2. Spectralassignments are summarized in Table 1.

TABLE 1 Wavenumber (cm⁻¹) Assignment 3,255 Includes the O—H stretchingvibration of the primary alcohol group and the N—H stretching vibrationsof the secondary aromatic amine and secondary amide groups. 3,200-2,700Includes ═C—H stretching vibrations of the aromatic ring andbenzimidazole group and the aliphatic C—H stretching vibrations.2,700-2,300 Includes the multiple NH⁺ stretching vibrations of thebenzimidazole 1:1 sulfate salt group. 1,673 C═O stretching vibrations ofthe secondary amide 1,653 group where the carbonyl group is subject todifferent environmental effects such as hydrogen bonding. 1,640-1,370Includes the C═C aromatic ring stretching vibrations, the C═C and C═Nstretching vibrations of the benzimidazole group, the O—H deformationvibration of the primary alcohol group and the aliphatic C—H deformationvibrations. 1,570 The CNH combination band of the secondary amide group.1,506 Includes the CNH bending vibration of the secondary aromatic aminegroup. 1,213 The aryl C—F stretching vibration. 1,189 The asymmetric SO₃⁻ stretching vibration of the benzimidazole 1:1 sulfate salt group.1,100-1,000 Includes the C—O stretching vibration of the primary alcoholgroup and the aryl C—Br stretching vibration. 1,011 The symmetric SO₃ ⁻stretching vibration of the benzimidazole 1:1 sulfate salt group.920-600 Includes the C—H wag vibrations and C═C ring bending vibrationsof the 1,2,4-trisubtituted aromatic ring and the benzimidazole group.  888 Includes the S—O(H) stretching vibration of the benzimidazole 1:1sulfate salt group.

Example 1A Preparation of the Hydrogen Sulphate Salt of Compound 1

Sulfuric acid (1.52 ml, 27.86 mmol) was added to a stirred suspension of6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxyethoxy)-amide (10 g, 0.0214 mol) (obtainable as describedin Example 10 of WO 03/077914, which is incorporated herein by referenceand as described below) in tetrahydrofuran (THF) (62 ml) and water (8ml) whilst maintaining a temperature of 10° C. or lower. The stirredmixture was heated to 65° C. and held for 30 minutes before filtering toremove any extraneous matter. THF (150 ml) was then added to the mixturemaintaining the temperature above 60° C. The mixture was then cooled to0-5° C. over approximately 2 hour. The resulting slurry was filtered,washed with THF (30 ml) and dried under reduced pressure at 50° C. untila constant weight was achieved, to give6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxyethoxy)-amide hydrogen sulfate (9.81 g, 0.17 mol, 82%yield) as an off white crystalline solid. The material was the same asthat produced in Example 1 above.

Example 1B Preparation of6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide Step A: 2,3,4-Trifluoro-5-nitro-benzoicacid

A 3 litre three neck round bottom flask was charged with 125 ml H₂SO₄.Fuming nitric acid was added (8.4 ml, 199 mmol) and the mixture gentlystirred. 2,3,4-Trifluorobenzoic acid (25 g, 142 mmol) was added in 5 gportions over 90 minutes. The dark brownish yellow solution was stirredfor 60 minutes at which time the reaction was complete. The reactionmixture was poured into 1 litre of an ice:water mixture and extractedwith diethyl ether (3×600 ml). The combined extracts were dried (MgSO₄)and concentrated under reduced pressure to give a yellow solid. Thesolid was suspended in hexanes and stirred for 30 min after which timeit was filtered to give 29 g (92%) of clean desired product as anoff-yellow solid: MS APCI (−) m/z 220 (M−1) detected.

Step B: 4-Amino-2,3-difluoro-5-nitro-benzoic acid

Ammonium hydroxide solution (˜30% in water) (35 ml, 271 mmol) was addedto a solution of 2,3,4-trifluoro-5-nitro-benzoic acid (15 g, 67.8 mmol)in 30 ml water at 0° C. with stirring. Upon completion of the ammoniumhydroxide addition, the reaction mixture was warmed to room temperaturewith stirring. After 2.5 hours, the reaction mixture was cooled to 0° C.and concentrated HCl was carefully added until pH of reaction mixturewas 0. The reaction mixture was diluted with water (30 ml) and extractedwith diethyl ether (3×50 ml). The combined organic extracts were dried(MgSO₄) and concentrated under reduced pressure to give 14 g (95%) ofpure desired product: MS APCI (−) m/z 217 (M−1) detected.

Step C: 4-amino-2,3-difluoro-5-nitrobenzoic acid methyl ester

A 2 M solution of tetramethylsilane (TMS) diazomethane in hexanes (6.88ml, 13.75 mmol) was added to a suspension of4-amino-2,3-difluoro-5-nitrobenzoic acid (2.00 g, 9.17 mmol) in 25 ml of4:1 Tetrahydrofuran (THF):MeOH at 0° C. under nitrogen atmosphere. Uponcompletion of addition, reaction mixture was warmed to room temperature.After 0.5 hours, excess TMS diazomethane was destroyed by the carefuladdition of acetic acid. The reaction was then concentrated underreduced pressure and dried in vacuo 1.95 g (92%) of pure desiredproduct: MS APCI (−) m/z 231 (M−1) detected.

Step D: 4-Amino-3-fluoro-5-nitro-2-phenylamino-benzoic acid methyl ester

4-Amino-2,3-difluoro-5-nitrobenzoic acid methyl ester (23.48 g, 101.1mmol) was suspended in xylenes (125 ml) and aniline (92 ml, 1011 mmol)was added. The reaction mixture was stirred at 125° C. for 16 hoursunder N₂. The reaction mixture was cooled to room temperature and solidsprecipitated out of solution. The solids were collected by filtrationand washed with xylenes and then diethyl ether. Recovered 22.22 g (72.78mmol) of yellow solid which was pure desired product. The filtrate wasconcentrated under reduced pressure, redissolved in methylene chlorideand flushed through a plug of silica gel eluting with methylenechloride. The desired fractions were concentrated under reduced pressureto give a brown solid which was triturated with diethyl ether to give5.47 g (17.91 mmol) of yellow solid which was pure desired product.Combined product yield was 27.69 g (90%). MS APCI (−) m/z 304 (M−1)detected.

Step E: 7-Fluoro-6-phenylamino-3H-benzoimidazole-5-carboxylic acidmethyl ester

4-Amino-3-fluoro-5-nitro-2-phenylamino-benzoic acid methyl ester (16.70g, 54.71 mmol), formic acid (250 ml, 6.63 mol) and 20% Pd(OH)₂/C (9.00g, 16.91 mmol) in ethanol (250 mL) were stirred at 40° C. for two hoursunder N₂ and then at 95° C. for 16 hours. The reaction mixture wascooled to room temperature and filtered through Celite rinsing withethyl acetate. The filtrate was concentrated under reduced pressure togive a yellow solid. The solid was triturated with diethyl ether to give13.47 g (86%) of the desired product as a tan solid. MS APCI (+) M/Z 286(M+1) detected; MS APCI (−) m/z 284 (M−1) detected.

Step F: 6-(4-Bromo-phenylamino)-7-fluoro-3H-benzoimidazole-5-carboxylicacid methyl ester

7-Fluoro-6-phenylamino-3H-benzoimidazole-5-carboxylic acid methyl ester(4.99 g, 17.51 mmol) was dissolved in N,N-dimethylformamide (275 ml).N-bromosuccinimide (3.15 g, 17.70 mmol) was added as a solid and thereaction mixture was stirred at room temperature under N₂. After 30minutes, the reaction mixture was quenched by the addition of aqueoussaturated sodium bisulfite solution. The reaction mixture was thenpoured into a separatory funnel, diluted with water and ethyl acetateand the layers separated. The aqueous layer was extracted with ethylacetate. The combined organic extracts were washed three times withwater, once with brine and then dried (Na₂SO₄) and concentrated underreduced pressure to yield 6.38 g (100%) of the pure desired product as atan solid. MS ESI (+) m/z 364, 366 (M+Br pattern) detected.

Step G:6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3H-benzoimidazole-5-carboxylicacid methyl ester

6-(4-Bromo-phenylamino)-7-fluoro-3H-benzoimidazole-5-carboxylic acidmethyl ester (6.38 g, 17.51 mmol), was dissolved inN,N-dimethylformamide (275 mL). N-chlorosuccinimide (2.36 g, 17.70 mmol)was added as a solid and the reaction mixture was stirred at roomtemperature under N₂ until the reaction is complete (5-6 days). Thereaction mixture was quenched by the addition of aqueous saturatedsodium bisulfite solution to give a suspension. The resulting solidswere collected by filtration, washed with water and diethyl ether anddried under reduced pressure to yield 6.07 g (87%) of the pure desiredproduct as a beige solid. MS ESI (+) m/z 398, 400 (M+Br pattern)detected.

Step H:6-(4-Bromo-2-chlorophenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid methyl ester and6-(4-Bromo-2-chlorophenylamino)-7-fluoro-1-methyl-1H-benzoimidazole-5-carboxylicacid methyl ester

A solution of6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3H-benzoimidazole-5-carboxylicacid methyl ester (150 mg, 0.38 mmol), iodomethane (28 μL, 0.45 mmol)and potassium carbonate (78 mg, 0.56 mmol) in dimethylformamide (1.5 mL)was stirred at 75° C. for one hour. The reaction mixture was dilutedwith ethyl acetate, washed with saturated aqueous potassium carbonate(2×), brine and dried (Na₂SO₄). Flash column chromatography (20:1methylene chloride/ethyl acetate) provided 56 mg (36%) of the moremobile6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid methyl ester as a white solid. ¹⁹F NMR (376 MHz, CD₃OD)—133.5 (s).MS APCI (+) m/z 412, 414 (M+, Br pattern) detected. Also isolated is 54mg (35%) of6-4(-bromo-2-chloro-phenylamino)-7-fluoro-1-methyl-1H-benzoimidazole-5-carboxylicacid methyl ester as a white solid. ¹⁹F NMR (376 MHz, CD₃OD)—139.9 (s).MS APCI (+) m/z 412, 414 (M+, Br pattern) detected.

Step I:6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid

6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid methyl ester (56 mg, 0.14 mmol) was dissolved into 2:1 THF/water (3mL) and NaOH (0.55 ml, 1.0 M aqueous solution, 0.55 mmol) was added.After stirring for two hours the reaction was reduced to one quarterinitial volume via rotary evaporation and the remainder diluted to 50 mlwith water. The aqueous solution was acidified to pH 2 by the additionof 1.0 M aqueous HCl and extracted with 1:1 tetrahydrofuran/ethylacetate (3×), dried (Na₂SO₄) and concentrated under reduced pressure toprovide 43 mg (79%) pure carboxylic acid as an off white solid. MS ESI(+) m/z 397, 398 (M+, Br pattern) detected.

Step J: 6-(4-Bromo-2chloro-phenylamino-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid(2-vinyloxy-ethoxy)-amide

6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2.00 g, 5.0 mmol), O-(2-vinloxy-ethyl)-hydroxylamine (0.776 g, 7.5mmol), HOBt (0.88 g, 6.5 mmol), triethylamine (1.61 mL, 2.3 mmol) andEDCl (1.3 g, 6.5 mmol) were dissolved in dimethylformamide (52 mL), andstirred at room temperature for 48 hours. The reaction mixture wasdiluted with ethyl acetate, washed with water (3×), saturated potassiumcarbonate (2×), saturated ammonium chloride (2×), brine, dried (Na₂SO₄)and concentrated under reduced pressure to an off-white solid.Trituration of the solid with diethyl ether provided 2.18 g (90%)desired product as an off-white solid. MS ESI (=) m/z 483, 485 (M+Brpattern) detected.

Step K:6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboyxlicacid (2-hydroxy-ethoxy)-amide

Hydrochloric acid (14 mL, 1.0 M aqueous solution, 14 mmol) was added toa suspension of6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboyxlicacid (2-vinyloxyethoxy)-amide (2.18 g, 4.50 mmol) in ethanol (50 mL) andthe reaction mixture allowed to stir for 24 hours. The reaction mixturewas concentrated to dryness by rotary evaporation and the solidspartitioned between 3:1 ethyl acetate/tetrahydrofuran and saturatedpotassium carbonate. The aqueous phase was extracted with 3:1 ethylacetate/tetrahydrofuran (3×), the combined organics dried (Na₂SO₄), andconcentrated to provide 2.11 g (100%)6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxyethoxy)-amide as an off-white solid. MS ESI (+) m/z 457,459 (M+, Br pattern) detected. ¹H NMR (400 MHz, MeOH-d₄) δ 8.26 (s, 1H),7.78 (s, 1H), 7.57 (d, 1H), 7.24 (dd, 1H), 6.40 (dd, 1H), 3.86 (s, 3H),3.79 (m, 2H), 3.49 (m, 2H). ¹⁹F NMR (376 MHz, MeOH-d₄)-133.68 (s).

Example 2 Investigation of the Physical Properties of the HydrogenSulfate Salt

The product of Example 1 was subject to the following tests to determineits physical properties.

Powder X-Ray Diffraction (PXRD)

All samples were run on a Bruker D5000 diffractometer. The X-ray powderdiffraction spectra were determined by mounting a sample of thecrystalline salt on Siemens single silicon crystal (SSC) wafer mountsand spreading out the sample into a thin layer with the aid of amicroscope slide. The sample was spun at 30 revolutions per minute (toimprove counting statistics) and irradiated with X-rays generated by acopper long-fine focus tube operated at 40 kV and 40 mA with awavelength of 1.5406 angstroms. The collimated X-ray source was passedthrough an automatic variable divergence slit set at V20 and thereflected radiation directed through a 2 mm antiscatter slit and a 0.2mm detector slit. The sample was exposed for 1 second per 0.02 degree2-theta increment (continuous scan mode) over the range 2 degrees to 40degrees 2-theta in theta-theta mode. The running time was 31 minutes and41 seconds. The instrument was equipped with a scintillation counter asdetector. Control and data capture was by means of a Dell Optiplex 686NT 4.0 Workstation operating with Diffract+software.

Data were collected over the range 2-theta 2-40°, in increments of2-theta 0.02° with 4 s per increment and are categorized in Table 2,with relative intensities derived from diffractograms measured withfixed slits.

TABLE 2 % Relative Intensity Definition 25-100 VS—Very Strong 10-25 S—Strong 3-10 M—Medium 1-3  W—Weak

The results of the scan are shown in FIG. 1, where the upper grey linerepresents the XRPD of the hydrogen sulfate salt of Compound 1 and thelower black line represent the free form. The most intense peaks,starting with most intense, are given in Table 3. The peak at 24.59° isparticularly strong.

TABLE 3 2 theta scale Relative intensity 24.59 VS 20.97 VS 23.99 S 27.65S 12.24 S 23.49 S 24.30 S 17.02 S 25.91 S 22.50 M

Persons skilled in the art of X-ray powder diffraction will realise thatthe relative intensity of peaks can be affected by, for example, grainsabove 30 microns in size and non-unitary aspect ratios, which may affectanalysis of samples. The skilled person will also realise that theposition of reflections can be affected by the precise height at whichthe sample sits in the diffractometer and the zero calibration of thediffractometer. The surface planarity of the sample may also have asmall effect. Hence the diffraction pattern data presented are not to betaken as absolute values (see Jenkins, R. & Snyder, R. L., “Introductionto X-Ray Powder Diffractometry”, John Wiley & Sons, 1996, for furtherinformation).

Example 3 In-Vivo Investigation Study of Salts Versus Free Base inDispersed Formulation

A study was performed in dogs to measure plasma levels of Compound 1 infasted dogs following administration of 150 mg free base equivalent oraldoses in 7.5 mL various dispersion formulations in pharmaceuticallyacceptable dispersing agents with Compound 1 contained as the free formor hydrogen sulfate salt.

Single doses of 150 mg were administered orally to fasted beagle dogsweighing 12-17 kg and about 2 to 6 years old on each of three dosingdays. Each dosing day was 1 week apart.

All formulations were prepared extemporaneously just prior to dosing byadding 7.5 mL of the appropriate dispersing solution via a 10 mLdisposable syringe, to vials containing 150 mg free base equivalents ofthe appropriate drug form, capping and vortex mixing for 30 seconds toform a dispersion.

The dispersion was removed from the vial using the disposable syringeand dosed to the animal via a gavage tube positioned into the stomach.The vials were rinsed twice by adding, for each of the two rinses, aseparate 15 mL aliquot of water (total rinse volume=30 mL) via a 20 mLdisposable syringe, capping, vortex mixing for 5 seconds, removing thewash solution from the vial using the disposable syringe and dosing tothe animal via the gavage tube.

Dogs were fed about 400 g of Special Diet Services Laboratory Diet Aeach day and allowed water ad libitum. Whole blood (2 mL) in lithiumheparin tubes were taken from the jugular vein immediately prior todosing and at 0.5, 1, 2, 3, 4, 5, 6, 8, 12, 18, 24, 36 and 48 hours. Theblood was centrifuged at 3000 rpm for 15 minutes and plasma was removedinto plain blood tubes and the plasma stored at −20° C. until analysis.

Plasma (50 mcL) was analyzed for Compound 1 concentration. Two dogs wereexcluded from the analysis as they had vomited just after dosing. Meanplasma concentration profiles for Compound 1 seen after oral dosing areshown in FIG. 3 where the line represented by ▴ illustrates aformulation which included the hydrogen sulfate salt of Compound 1, andthe line represented by x shows the results of Compound 1 free base inthe same formulation.

It appears that formulation changes had a relatively small affect onexposure (results not shown). However when Compound 1 was dosed as thehydrogen sulfate salt, a substantial approximately 4 to 8 fold increasein exposure was produced.

The foregoing description is considered as illustrative only of theprinciples of the invention. Further, since numerous modifications andchanges will be readily apparent to those skilled in the art, it is notdesired to limit the invention to the exact construction and processshown as described above. Accordingly, all suitable modifications andequivalents may be resorted to falling within the scope of the inventionas defined by the claims that follow.

The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of stated features, integers,components, or steps, but they do not preclude the presence or additionof one or more other features, integers, components, steps, or groupsthereof.

1. A crystalline hydrogen sulfate salt of6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide, wherein said hydrogen sulfate salt of6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide has an X-ray powder diffraction patternwith at least one specific peak at about 24.59°.
 2. The crystallinehydrogen sulfate salt of6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide according to claim 1, wherein saidhydrogen sulfate salt of6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide has an X-ray powder diffraction patternwith specific peaks at about 2-theta equal to 24.59°, 20.97°, 23.99°,27.65°, 12.24°, 23.49°, 24.30°, 17.02°, 25.91° and 22.50°.
 3. Thecrystalline hydrogen sulfate salt of6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide according to claim 1, which has a powderX-ray diffraction pattern substantially the same as the X-ray powderdiffraction pattern shown in FIG. 1.